Quality of life during one-year observation of patients with idiopathic pulmonary fibrosis awaiting lung transplantation.

The aim of the study was to evaluate the quality of life and its changes in patients with idiopathic pulmonary fibrosis (IPF), waiting for lung transplantation. Sixteen IPF patients, who qualified for lung transplantation, and 14 chronic obstructive pulmonary disease (COPD) patients on long term oxygen therapy, taken as a reference group, were assessed. The quality of life was estimated twice, using an SF-36-questionnaire, at the time of qualification for lung transplantation and 12 months later. The IPF and reference patients had similarly profound disturbances in lung function and arterial blood gas content. The IPF patients had a statistically greater score in their physical functioning (PF; 45 vs. 18), the role of limitations due to physical problems (RP; 43 vs. 11), social functioning (SF; 58 vs. 30), and the role of emotional limitations in everyday life (RE; 65 vs. 12) than the reference ones. After a year's observation, there were no differences in SF-36 questionnaire results, except for the role of limitations due to physical problems (RP). A correlation was noted between spirometry and blood gas results and SF-36 questionnaire results in IPF patients qualified for lung transplantation. There was a positive correlation between the partial pressure of oxygen (PaO(2)), on one side, and mental and general health, on the other. There also were positive correlations between FEV1 and SF and FEV1 and the level of bodily pain (BP). We conclude that the SF-36 questionnaire is a sensitive tool to assess the quality of life in IPF patients qualified for lung transplantation. The information gained can help assess the severity of the disease, clinical symptoms, and functional impairment in these patients.

The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells.

The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Three R's of bacterial evolution: how replication, repair, and recombination frame the origin of species.

The genetic diversity of bacteria results not only from errors in DNA replication and repair but from horizontal exchange and recombination of DNA sequences from similar and disparate species as well. New individuals carrying adaptive changes are thus being spawned constantly among the population at large. When new selection pressures appear, these are the individuals that survive, at the expense of the general population, to forge new populations. Depending on the severity and uniqueness of the selection pressure, this could lead to new speciation. It is becoming more and more evident that, as nucleotide sequences of numerous loci from many bacterial strains continue to amass, horizontal transfer has played a key role in configuring the Escherichia coli chromosome. Here, we examine views, both old and new, for the role of recombination in the evolution of bacterial chromosomes. We present novel phylogenetic evidence for horizontal transfer of three genes involved in DNA replication and repair (mutS, uvrD, and polA). These data reveal a prominent role for horizontal transfer in the evolution of genes known to play a key role in the fidelity of DNA replication and, thus, ultimate survival of the organism. Our data underscore that recombination plays both a diversifying and a homogenizing role in defining the structure of the E. coli genome.

Fidelity of replication of repetitive DNA in mutS and repair proficient Escherichia coli.

Replication fidelity is not constant among strains within a species or at all genetic loci within a genome. Altered fidelity of replication may affect patterns of pathogenesis and the evolution of these strains. We have been studying replication fidelity in Escherichia coli, both in laboratory attenuated strains and in food-borne pathogens. To understand the altered patterns of mutagenesis at the molecular level, we used a shuttle vector plasmid with a tRNA mutational marker gene which had been altered to include homopolymeric runs of five, seven and nine [G:C] pairs, as well as non-repetitive DNA. Replication of the plasmid in mutS strains resulted in a 20-fold increase in mutant progeny plasmids. The mutations were almost all (>90%) frameshift mutations, while base substitution mutations were rare. Most mutations were insertions or deletions of one or two [G:C] pairs in the longest homopolymeric runs. Larger deletions (5 to >70bp), also targeted to the repetitive sequence, were likewise common. Mutations increased exponentially with the length of the homopolymeric run. These patterns of mutation, including unexpectedly high levels in repair proficient strains, led to an examination of the E. coli K-12 genome for homopolymeric DNA. This sequence motif was found to be rare, particularly in genes and open reading frames. Amino acid homotrimers were found to avoid usage of homopolymeric codons, even when they are preferred among synonymous codons in E. coli. There appears to be active selection against tandem direct nucleotide repeats in the E. coli genome, correlated with the inability of the organism to accurately replicate such sequence.

Phylogenetic evidence for horizontal transfer of mutS alleles among naturally occurring Escherichia coli strains.

mutS mutators accelerate the bacterial mutation rate 100- to 1,000-fold and relax the barriers that normally restrict homeologous recombination. These mutators thus afford the opportunity for horizontal exchange of DNA between disparate strains. While much is known regarding the mutS phenotype, the evolutionary structure of the mutS(+) gene in Escherichia coli remains unclear. The physical proximity of mutS to an adjacent polymorphic region of the chromosome suggests that this gene itself may be subject to horizontal transfer and recombination events. To test this notion, a phylogenetic approach was employed that compared gene phylogeny to strain phylogeny, making it possible to identify E. coli strains in which mutS alleles have recombined. Comparison of mutS phylogeny against predicted E. coli "whole-chromosome" phylogenies (derived from multilocus enzyme electrophoresis and mdh sequences) revealed striking levels of phylogenetic discordance among mutS alleles and their respective strains. We interpret these incongruences as signatures of horizontal exchange among mutS alleles. Examination of additional sites surrounding mutS also revealed incongruous distributions compared to E. coli strain phylogeny. This suggests that other regional sequences are equally subject to horizontal transfer, supporting the hypothesis that the 61.5-min mutS-rpoS region is a recombinational hot spot within the E. coli chromosome. Furthermore, these data are consistent with a mechanism for stabilizing adaptive changes promoted by mutS mutators through rescue of defective mutS alleles with wild-type sequences.

Promiscuous origin of a chimeric sequence in the Escherichia coli O157:H7 genome.

A novel sequence of 2.9 kb in the intergenic region between the mutS and rpoS genes of Escherichia coli O157:H7 and closely related strains replaces a sequence of 6.1 kb in E. coli K-12 strains. At the same locus in Shigella dysenteriae type 1, a sequence identical to that in O157:H7 is bounded by the IS1 insertion sequence element. Extensive polymorphism in the mutS-rpoS chromosomal region is indicative of horizontal transfer events.

Detection of mutator subpopulations in Salmonella typhimurium LT2 by reversion of his alleles.

Defects in the methyl-directed mismatch repair lead to both the hypermutability phenotype and removal of a barrier to genetic exchange between species. Mutator bacteria carrying such defects occur frequently among bacterial pathogens, suggesting that subpopulations of mutators are contained within pathogen clones and give rise to the genetic variants that are acted upon by selective forces to allow survival or successful infection. We report here on the detection of the mutator subpopulation in Salmonella typhimurium and determination of its frequency in laboratory cultures. The analysis involved screening for mutators among revertants of S. typhimurium histidine auxotrophs selected for the His+ phenotype, since the frequency of mutators is expected to be increased in the selected mutant population they helped to spawn. The increases in spontaneous reversion of histidine mutations were first measured in isogenic strains carrying mismatch repair-defective mutH, mutL, mutS, or uvrD alleles, relative to their mismatch repair-proficient counterparts. Screening for the mutator phenotype in nearly 12,000 revertants of repair-proficient strains carrying his mutations highly stimulated for reversion in mutator backgrounds, the base substitution in hisG428 and frameshift in hisC3076, yielded five mutator strains (0.04%). The his+ reversion mutations contained within the newly-arisen mutator strains were characteristic of the predominant nucleotide changes expected in such mutators, as assessed by comparison with the spectra for reversion events in wild-type and mismatch correction-defective backgrounds. The results show that subpopulations of mutators, residing in normal populations at a finite frequency, can be culled from the culture by strong selection for a required phenotype. We calculate that the frequency of mutators in the unselected population of S. typhimurium is 1-4x10-6, an incidence 10-fold lower than that expected based on studies of laboratory cultures of Escherichia coli.

Specificity of base substitution mutations induced by the dietary carcinogens 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in Salmonella.

The base pair substitution mutational profiles induced by the heterocyclic amine cooked food mutagens PhlP and IQ in Salmonella typhimurium strains TA100 and TA1535 were determined by colony hybridization analysis. Both PhlP and IQ induced predominantly GC-->TA transversions in strain TA100 (rfa,delta uvrB/pKM101) with a pronounced preference for the second codon position (CCC--> CAC; 72% of total). PhlP also reverted strain TA1535 (rfa, delta uvrB) efficiently at concentrations similar to those required for strain TA100. In contrast to the PhlP-induced mutational profile observed in strain TA100, in strain TA1535 PhlP induced exclusively GC-->AT transitions at the second codon position (CCC-->CTC; 96-99% of total). Base substitution mutagenesis induced by heterocyclic amines related to PhlP is generally SOS-dependent, requiring the presence of plasmid pKM101 in Salmonella hisG46 strains. Thus, the SOS dependent reversion of S. typhimurium strain TA100 probably reflects error-prone lesion bypass at the major PhlP- guanosine adduct at the C-8 position. The GC-->AT transition mutations induced by PhlP in strain TA1535 appear to be SOS-independent, however, suggesting that these mutations may arise from the formation of PhlP-DNA adducts other than the replication-blocking C8-dG lesion.

Identification of genetic variants of hepatitis A virus.

Although detection of hepatitis A virus (HAV) has been greatly aided by the development of polymerase chain reaction (PCR) technology, identification of genetic variants requires sequencing PCR products, which necessarily limits the length of the HAV genome (typically 2%) that can be analyzed. From a regulatory standpoint, identification of the specific strain detected by PCR is a prerequisite not only to overrule contamination of test samples in the diagnostic laboratory, but also to possibly locate the origin of the virus detected by PCR. We explored alternatives to sequencing PCR products to achieve these goals. The findings indicate that restriction fragment length polymorphism (RFLP) analysis of PCR products from two noncontiguous regions of the HAV genome encompassing 765 nucleotides (approximately 10% of the genome) by the restriction endonucleases HinfI and AluI, which cut frequently within the HAV genome, can distinguish the common tissue culture adapted strains of HAV from stool isolates. The resolution can be greatly enhanced by combining single strand conformation polymorphism (SSCP) analysis with restriction enzyme digestion, when most of the seventeen strains analyzed could be identified.

Detection and characterization of the fimA gene of Escherichia coli O157:H7.

An expected 850-bp DNA fragment containing fimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains of Escherichia coli using the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13 HinP1 and four Sau961 restriction profiles among these 39 E. coli strains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared by E. coli O157:H7, O157:H- and a few O55 serotype strains. DNA sequence analysis of the fimA region demonstrated that E. coli O157:H7 strain 933 and O157:H- strain E32511 contained identical DNA sequences that were distinct from other E. coli strains, especially a 16-bp sequence 5' to fimA that was conspicuously absent only in E. coli O157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from all E. coli O157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detect E. coli strains of the O157:H7 serotype.

Adaptive mutation and slow-growing revertants of an Escherichia coli lacZ amber mutant.

We have studied revertants, selected on lactose minimal agar medium, of the Escherichia coli lacZam strain that was first used by Cairns and his colleagues to demonstrate the phenomenon of "adaptive mutation." We have found, by performing appropriate reconstruction studies, that most of the late-arising Lac+ revertants of this lac amber strain (appearing as colonies in 3-5 days) are slow-growing ochre suppressor mutants that probably existed in the culture prior to plating and cannot, therefore, be classified as "adaptive." The appearance of a small number of fast-growing, late-arising Lac+ revertants may result from residual cell growth and turnover or from phenomena related to the fact that the lacZam mutation in strain SM195 is carried on an F' plasmid. Thus, the appearance of late-arising revertants in this lacZam system does not provide convincing evidence that selective conditions specifically increase the rate of occurrence of favorable mutations.

High mutation frequencies among Escherichia coli and Salmonella pathogens.

Here it is reported that the incidence of mutators among isolates of pathogenic Escherichia coli and Salmonella enterica is high (over 1 percent). These findings counter the theory, founded on studies with laboratory-attenuated strains, that suggests mutators are rare among bacterial populations. Defects in methyl-directed mismatch repair underlie all mutator phenotypes described here. Of nine independently derived hypermutable strains, seven contained a defective mutS allele. Because these mutant alleles increase the mutation rate and enhance recombination among diverse species, these studies may help explain both the rapid emergence of antibiotic resistance and the penetrance of virulence genes within the prokaryotic community.

Diagnostic potential of sefA DNA probes to Salmonella enteritidis and certain other O-serogroup D1 Salmonella serovars.

Salmonella enteritidis thin fimbriae, SEF14, were found to be restricted to S. dublin and the predominantly poultry-associated members of the Salmonella O-serogroup D1, S. enteritidis, S. berta, S. gallinarum and S. pullorum, when tested by Western and ELISA analysis from among 90 Salmonella isolates of 42 serovars, as well as from members of several related genera of the Enterobacteriaceae. These five serovars and a single isolate of S. typhi (D1) were also detected by hybridization of genomic DNA from 732 Salmonella isolates of 117 serogroups to gene probes derived from the S. enteritidis sefA (fimbrin gene), sefB (chaperone) or sefC (outer membrane protein) genes encoding proteins involved in SEF14 biosynthesis. None of 250 Enterobacteriaceae or 27 other eubacterial isolates tested hybridized to the sef probes. The sefA, sefB and sefC genes were amplified from these six Salmonella serovars by PCR using primer pairs designed from sefA, sefB or sefC of S. enteritidis. DNA sequencing of sefA genes from these five serovars indicated limited sequence variability among sefA genes and recognition of individual base pairs which could potentially differentiate certain strains of S. enteritidis, S. dublin and S. gallinarum.

Molecular analysis of Salmonella hisG428 ochre revertants for rapid characterization of mutational specificity.

The Salmonella typhimurium tester strains TA104 and TA102 were developed primarily to aid in the detection of oxidative mutagens and other agents that react preferentially with AT base pairs. Reversion of prototrophy of strains harboring the hisG428 ochre allele can occur by (i) any of seven single base substitutions or (ii) several tandem double base substitutions at the ochre codon, (iii) in-frame deletions removing all or part of the ochre codon or (iv) mutations at several distinct tRNA extragenic suppressor loci. We have used allele-specific oligonucleotide probes and DNA sequence analysis to characterize 625 revertants of strain TA104 (hisG428, rfa, DeltauvrB/pKM101) arising spontaneously or after treatment with methyl methane-sulfonate, glyoxal, streptonigrin or angelicin with UVA radiation. The reversion profiles obtained from these analyses distinguished readily each of the mutagen-treated populations from one another and from spontaneously derived revertants. Both GC and AT base pair-specific revertants were observed. Molecular analyses of S. typhimurium hisG428 revertants permitted rapid assessment of base pair substitution specificity of mutagens, especially the detection of AT base pair substitutions not recovered in strains carrying the complementary hisG46 allele.

Salmonella typhimurium LT7 and LT2 strains carrying the imp operon on colIa.

The imp operon is carried on a transmissible plasmid, ColIa, in original isolates of Salmonella typhimurium LT7. LT2 strain recipients of F' factors from LT7 strains harboring ColIa can acquire ColIa and imp under nonselective conditions. Thus, S. typhimurium LT2 strains that have received plasmids by conjugal transfer from LT7 strains might be inadvertently harboring ColI factors.

Reversion profiles of coolwhite fluorescent light compared with far ultraviolet light: homologies and differences.

General Electric and Sylvania 15 W coolwhite fluorescent lamps emit roughly 6% of their total irradiance as light in the UV spectrum. Illumination of sensitive Salmonella tester strains results in both lethal and mutagenic activities. In contrast, comparable Philips lamps emit lower levels of UV light, especially UVB, and exhibit no detectable lethal or mutagenic effects. The spectra of mutations induced by General Electric coolwhite lamps in histidine-requiring base substitution mutants hisG46 and hisG428 ("reversion profiles") resemble mutagenesis by far UV light (UVC) and differ quite markedly from the spectra of mutations that occur spontaneously. Coolwhite and UVC reversion profiles are not identical, however. The percentage of C to A transversion mutations induced in hisG46 are elevated over those found after UVC treatment, and a strong bias for one particular class of tandem base substitutions (TAA-->TGT) prevails after treatment of hisG428 with coolwhite light, a bias not observed with UVC. Increased attention needs to be given to minimization of exposure to UV light from fluorescent lamps commonly used in homes and workplaces.